This project studies physiological and cellular aspects of neuronal calcium signaling, with long-range emphasis on dendrites and dendritic spines of central nervous system neurons. Recent work has focused on Ca regulation in bullfrog sympathetic neurons because these large, spherical cells are excellent models for revealing intracellular details of Ca movements. Sympathetic neurons respond to depolarizing stimuli with a rise in cytosolic free Ca concentration ([Ca2+]i) that is initiated by Ca2+ entry through voltage-gated channels but also modulated by the activity of intracellular Ca stores. We have now studied the role of one major intracellular store, the mitochondrion, in defining the time course of [Ca2+]i during and after membrane depolarization. We had earlier shown that in these neurons depolarization-induced increases in [Ca2+]i are accompanied by large, graded, and reversible elevations in total mitochondrial calcium concentration ([Ca]m). Interestingly, these increases were spatially heterogeneous both among and within individual mitochondria. Therefore, we have characterized the distribution and chemical form of intramitochondrial Ca by using a novel high-resolution technique, spectrum imaging by electron energy loss spectroscopy, to map, at better than 10-nm resolution, the spatial distribution of intracellular Ca. Within single mitochondria of depolarized cells, Ca was concentrated in small (<10 nm) phosphorus-rich inclusions; their high Ca content suggests that they are quasi-crystalline. These inclusions likely reflect a high-capacity mechanism of intramitochondrial Ca sequestration. In addition to intra-organelle heterogeneity, the number and size of inclusions was higher in peripheral than in internal mitochondria, so that depolarization-evoked elevations in [Ca]m showed a radial dependence on distance from the plasma membrane. The distribution of [Ca]m is consistent with our diffusion simulations based on established models of Ca2+ transport. Persistence of the [Ca]m gradient during maintained depolarization implies that mitochondria have a memory, retaining a record of early spatial differences in [Ca2+]i long after free ion gradients have dissipated. Work is now underway to characterize the role of a second major Ca-regulating organelle, the endoplasmic reticulum, and to define its spatio-temporal interactions with the mitochondrial Ca pool. - calcium/ hippocampus/ dendrites/ sympathetic neuron/ endoplasmic reticulum/ mitochondria/ synapse/ x-ray microanalysis/ electron energy loss spectroscopy